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Improved Early detection of Crop Disease


<OL> <LI> Hybridize GN1-GFP and PR1-GFP transgenic plants that have been produced carrying an enhancer and characterize the fluorescence response. <LI> Perform microarray analysis of arabidopsis against pathogens for canola and arabidopsis using new gene chips. <LI> Hydroponics assay of transgenic plants with photonic tools. <LI> Begin work on creating a synthetic promoter for pathogen sensing that can be fused with GFP. </ol> The long-term goal of the research is to produce a crop-based phytosensor platform with which to monitor the onset and spread of crop diseases for the purpose of early intervention.

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NON-TECHNICAL SUMMARY: Current plant pathogen detection relies on dete tion of symptoms. Needed is a system that detects plant diseases presympomaticlly. The purpose of this study is to provide the reserch base designed to create an early detection system for the detection of plant pathogens.

<P>APPROACH: We will create a molecular-based platform that can someday be deployed potentially in all crop species to sense plant pathogens before they cause disease. This will be accomplished using pathogen-responsive promoters fused to the gene encoding green fluorescent protein (GFP). When pathogen(s) land on leaves, the plant will sense it and produce GFP, which has a unique spectral signature and can be remotely sensed. Initially, we will use a promoter of pathogenesis responsive genes (PR) fused to GFP in Canola. Microarray analysis will be conducted to identify responsive genes to three economically important diseases. A highly controlled hydroponics/controlled environment system will be used to evaluate the physiological characteristics and general `fitness' of the transgenic lines and hybrids produced. An important consideration throughout the project will also be to work on photonic devices that can be used to detect and measure changes of GFP in plants. Two new, state of the art instruments will be evaluated. Both are capable of being interfaced with the global positioning system to remote sensing instruments deployed from aircraft or satellites.
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PROGRESS: 2003/07 TO 2006/06<BR>
The project has several objectives: Objective 1. Construct a binary vector containing a translational fusion of the Arabidopsis thaliana PR-1a promoter, and mGFP5-ER, and transform this construct into tobacco as a model plant and proxy for soybean. Accomplishment: The objective was completed as written. See publications below. Objective 2. Characterize transgenic plants for their spectral responses to a suite of plant pathogens in growth chamber/greenhouse studies. Certain pathogens that cause GFP spectral signatures will be tested against plants in the field. Accomplishment: A partial screen was completed. About the time that the work was beginning, spill-over in additional regulations were put in place restricting agents that might be used in terrorist activities, e.g., plant pathogenic bacteria and fungi. It made acquiring pathogens difficult (we only just now received the USDA permit! And it made field testing against our plant of interest impossible. Nonetheless, the work has been published (see above), and we are now continuing the screen in an acceptable form (unpublished data). Objective 3. Transform soybean to produce a pathogen sentinel crop. Accomplishment: This experiment did not work, although due diligence was expended. Instead, we focused on the production of transgenic canola as an oilseed crop and improved the transformation procedure (Cardoza and Stewart 2003). Objective 4. Use GFP in a promoter/enhancer trapping construct to perform T DNA insertion mutagenesis experiments in tobacco to identify, characterize, and clone elements that are responsive to diseases not PR-1a inducible. Accomplishment 4: A screen was performed in approximately 1000 lines. The work was part of a graduate student thesis cited below (Kooshki 2004). Objective 5. Hydroponics assay of transgenic plants with photonic tools. Accomplishment: We learned much about how fluorescent protein dynamics in planta. The most precise way to do this was via hydroponic growth under strictly controlled conditions. Coupled with the detection methods for fluorescent proteins in planta, we made great strides toward reduction to practice of the technology. See the several publications with Halfhill as first author). Objective 6. Troubleshoot and perfect photonic measurements using fluorescence tools. Accomplishment: The long-term goal of the research is to produce a crop-based biosensor platform with which to monitor the onset and spread of crop diseases for the purpose of early intervention. A large technological hurdle is the imaging of a reporter gene in plants. The critical contribution of this grant was the expression of fluorescent proteins and their detection. See the publication list below for the accomplishments. Objective 7. Production of synthetic pathogen-inducible promoters fused to reporter genes. Accomplishment: This work has been largely completed but the grant terminated prior to publication, and the post-doc working on the project had to be released. Between synthetic promoter technology development and the perfection of using GFP in plants, however, more was accomplished than initially thought possible.
IMPACT: 2003/07 TO 2006/06 <BR>
The research is leading to the development of an early detection system for plant diseases that centers on expression of a detectible protein in leaves. The system will contribute to lower pesticide applications and less potential, negative impacts on the environment.

Rufty, Tom
North Carolina State University
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